The objective of the study was to assess pH measurements between offal organs of different species and the association between pH taken 4 h post-slaughter and different predictor variables in the liver and lungs. A linear regression analysis was conducted on selected variables to identify the main predictors and their interactions affecting the pH of meat 4 h post-slaughter. In an increasing order of magnitude during winter, the pH achieved at 16 h - 36 h post-slaughter in springbok heart, liver, spleen, kidney and lungs was significantly (p < 0.05) higher than pH 6.0. The pH attained in springbok carcasses was (p < 0.05) below 6.0, whilst no significant differences were observed from the regulatory reference (pH 6.0) in the heart. There was a positive association between the pH of game meat 4 h post-slaughter and liver congestion. The pH of game meat 4 h post-slaughter increased by 0.11 units (p < 0.05) per millilitre increase in liver congestion and decreased by 0.04 units (p < 0.05) per minute increase in the shooting-to-bleeding interval, irrespective of the species. The lack of a statistically significant association between some selected variables and pH changes in this study suggested that either the factors may have a small effect which is only detectable with large data-sets and/or the effect may be modified by other unidentified factors. As some of the offal organs had final pH readings above 6.0, alternative measures are required to inactivate certain endogenous pathogens in edible wild game offal sourced from endemic areas.

Introduction

Edible offal offers a range of foods which are nutritiously attractive, especially in developing countries. The edible offal is highly prized in South East Asia and Africa, whilst demand is variable and low in Australia and the USA, respectively (Fatma & Mahdey 2010). In slaughtered animals, edible offal contributes 33% of the edible material (Aduku et al. 1991; Pearson & Dutson 1988). It is estimated that about 75% of the emerging human infectious diseases arise from animal reservoirs (Allen et al. 2012), of which Rift Valley fever (RVF) is placed third on the list of the 17 most dangerous animal threats after foot and mouth disease (FMD) and influenza (Mandell & Flick 2011). A pH value below 6.0 is commonly used to destroy the most dangerous animal pathogens and ensure the safety of livestock products. Microorganisms have optimum, minimum and maximum pH for growth in different foods as a result of the interactions between the pH and other factors which either promote or inhibit microbial growth. In general, microbes do not grow, or else grow very slowly, at pH below 4.6, although there are exceptions. The pH of meat is important for good carcass quality and for inactivating viral and bacterial animal microbes, a number of which are endemic in southern Africa (European Commission [EC] 2001; Falenski et al. 2010; Fatma & Mahdey 2010).

A serious zoonotic livestock disease, such as RVF, poses a significant public health threat and can have serious consequences. It is presumed that certain wildlife species (African buffalo, lesser kudu, black rhinoceros, white rhinoceros, lion and impala) may carry the RVF virus as reservoirs between outbreaks and that wildlife meat that is not properly handled may transmit the RVF virus to humans during that period (American Veterinary Medical Association 2010; Evans et al. 2008). Preferred samples for isolation of Bunyaviridae (genus Phlebovirus), the cause of RVF, are blood (preferably collected in an anticoagulant-containing vessel during the febrile stage of the disease) or liver, spleen and brain tissues of dead animals and organs of aborted foetuses (World Organisation for Animal Health [OIE] 2012a). RVF virus is a relatively unstable agent and at its optimum pH range of 7.4-8.0 and with 2% serum, it has a half-life of 6.8 days at 0 °C and 1.5 h at 37 °C. No references were found on the persistence of RVF virus in carcasses or muscle during the conduct of this study, but the virus would be expected to disappear quickly from the carcass as the pH dropped with rigor mortis (Scott 2003).

Inactivation of the FMD virus in skeletal muscle and heart muscle occurs rapidly after animal death as a result of lactic acid formation which accompanies rigor mortis and causes the pH to drop to levels between 5.5 and 6.0. The inactivation rate for FMD virus at pH 6.0 is 90% per minute, whilst at pH 5 it is 90% per second (Bachrach et al. 1975). In skeletal muscle kept at 4 °C, the FMD virus is regarded as completely inactivated within 48 h, although infectivity may remain for over 4 months in lymph nodes, clotted blood, bone marrow and viscera where the virus is protected from maturation-induced pH changes (Blackwell 1984). The FMD virus is most stable around neutral pH and is sensitive to even mild acidity. Although temperatures above 50 °C destroy most FMD virus infectivity, it is likely that a small proportion of particles are relatively resistant to the effects of heat and pH (Hyslop 1970; MacDiarmid & Thompson 1997). However, the muscle pH of animals slaughtered in a febrile state may not drop below 6.0; hence, some FMD virus may survive in muscle tissue of such animals up to 96 h post-slaughter (MacDiarmid & Thompson 1997). Caution in extrapolating findings between species is recommended because of a reported case of highly myotropic FMD virus surviving beyond 72 h in the carcass of impala when the pH had dropped to 5.6 (Bengis & Veary 1997; Scott 2003).

Brucella melitensis and Brucella abortus have been detected at varying counts in edible offal (Fatma & Mahdey 2010). Brucella spp. bacteria survival time in chilled meat seems extremely short, except in frozen carcasses where the organism can survive for years (EC 2001). These bacteria often survive desiccation and can survive freezing and near-freezing temperatures for over two years (Iowa State University 2011). The number of Brucella organisms per gram of muscle is normally small and rapidly decreases with the drop in meat pH (EC 2001; El-Nesser et al. 2007; Sadler 1960). The survival period of B. abortus in yoghurt at pH 3.60 and 3 °C is 1 day. The optimal pH range of Brucella is between pH 6.6 and 7.4 at 37 °C, whilst the maximum pH is 8.4 and the minimum is 4.1 (Falenski et al. 2010; International Commission on Microbiological Specifications for Food 1996; Lerche & Entel 1959; Zobell & Meyer 1932).

Within the animal-human interface, transmission of brucellosis and RVF virus as zoonotic food-borne pathogens frequently depends on factors such as food consumption habits and level of processing (El-Nesser et al. 2007; Fatma & Mahdey 2010; Michael et al. 2011; United States Department of Agriculture 2011). At present, the OIE Terrestrial animal health code (OIE 2012b) has no recommendations to veterinary authorities concerning the importation of edible offal from countries that are not free from Brucella, FMD virus and RVF virus. However, the animal and public health requirements for wildlife meat destined for export are sometimes stipulated by the importing country in the import permit. Some importing countries require only carcasses to be submitted to veterinary maturation at a temperature above +2 °C for at least 24 h. The intended goal of this intervention is to attain an electronically measured pH reading from the middle of the longissimus dorsi below pH 6.0 (Bengis & Veary 1997; EC 2010; Pharo 2002). Deliberate and natural pH reduction in meat is the cheapest and commonest intervention that can be applied to meat and meat products to destroy or control pathogens to ensure public health and food security (Institute of Food Technologists [IFT] & Federal Drug Administration [FDA] 2003).

Materials and methods

Measurement of pH

The pH and temperature were measured using a testo model 205 (Testo AG, Lenzkirch, Germany) temperature and pH meter with automatic temperature compensation. The selection of farms included in this study was based on application by owners for game harvesting for commercial purposes. For eviscerated carcasses with skin on, meat pH was measured 16 h - 34 h post-slaughter by inserting the probe in the middle of the longissimus dorsi, whilst for edible offal organs the pH meter probe was inserted into the organ. Calibration of the testo meter was achieved using a standard calibration solution at the beginning of the measurements and after every five readings.

Winter night harvesting

The springbok (Antidorcas marsupialis) were harvested between July and August 2010. Average night temperatures ranged from 0 °C to 17 °C. Average maximum and minimum humidity was 50% and 7%, respectively. Relative humidity was measured using a hygrometer (Hygrocheck; Hanna Instruments, Johannesburg, South Africa) and verified by readings from Intellicast (http://www.intellicast.com/Global/Humidity.aspx). The pH measurements were derived from five consignments of springbok harvested on different days and delivered to the abattoir at a minimum of 10 carcasses per group. In total, 55 harvested springbok carcasses and organs (liver, lung, heart, spleen and kidney) were selected from visually inspected and passed carcasses.

Summer daylight harvesting

The harvesting was conducted on one farm and the random effects of the three harvesting camps on the farm were assumed to be normally distributed. Species under consideration were eland (Taurotragus oryx), red hartebeest (Alcelaphus buselaphus), springbok and kudu (Tragelaphus strepsiceros). All red hartebeest were males except for one female, springbok were females except for two males, kudu were females and eland were males except for one female. All animals were mature. The game harvesting was conducted during the day and during the summer month of February. Average day temperatures ranged from 22 °C to 31 °C. A progressive decrease in the relative humidity from morning to evening was observed, with a maximum humidity of 84% at approximately 07:20 and a minimum humidity of 23% at approximately 17:00. All harvested game included in this study were healthy. The shooting position was consistently the upper neck and head. In springbok, evisceration of the stomachs and intestines (white offal) was performed 1.25 h after bleeding, whilst in the hartebeest, eland and kudu, it was performed at bleeding soon after shooting (Van Schalkwyk et al. 2011).

All harvested animals were given a visual body condition (Table 1). The selection of variables was based on previous postmortem observations during game harvesting inspections. The time (in minutes) taken chasing the identified wild animal until shooting, as well as the time from shooting to severing blood vessels (carotid and jugular) in the neck below the jaw line by means of a deep cut with a sharp disinfected knife, was recorded. The liver and lung temperature and pH were recorded 4 h after the blood vessels in the neck were severed, soon after the red offal (heart, liver, kidney, spleen, lungs, trachea and oesophagus) evisceration. Liver and lung congestion was scored using the rating system of the quantity (mL) of blood collected in a graduated tube from a longitudinal cut across the whole organ followed by organ squeezing for 2 min. The cut was made on the gastric surface of the liver and dorsal surface of the lung. Livers and lungs that had blood on the incision line after 2 min that did not flow into the tube were given a score of 0.09 mL.

Statistical analysis

For winter night harvesting, the Student's f-test and one-way analysis of variance (ANOVA) using the Statistical Analysis Systems (SAS) version 9.1.3 (2007) was used to evaluate pH measurements between organs, and between organs and reference pH value, with significance at p < 0.05 andp < 0.01, respectively. Data are expressed as mean ± s.d.

For the summer daylight harvesting, all analysis was conducted using Stata version 10 (2007). Descriptive statistics describing all variables were used on the data. Linear regression was used to evaluate the association between pH after 4 h (outcome) and the predictor variables. Univariate analyses between pH after 4 h and all the potential predictors (time from chasing to shooting, time from shooting to bleeding, offal temperature at 4 h post-slaughter, liver congestion, lung congestion, body condition score, environmental temperature, humidity, species, liver and lung pH at 4 h post slaughter) were first evaluated to detect those variables that were statistically significant. This was followed by fitting multivariable models evaluating the association between pH after 4 h and the identified predictors of interest and controlling for potential confounder variables. A value of p < 0.05 was considered significant. Model diagnostics were conducted to evaluate the assumptions of homoscedasticity and normality of residuals.

Ethical considerations

Harvesting was performed by approved game harvesting teams registered by the Ministry of Environment and Tourism and the Directorate of Veterinary Services of Namibia. Harvesting was conducted following international ethical guidelines stipulated in Nature Conservation Ordinance (No. 4 of 1975) (Territory of South West Africa 1975), as amended in the Guidelines for the harvesting of game for meat export (Van Schalkwyk & Hoffman 2010). The harvesting process was monitored by officials from the two abovementioned institutions to ensure adherence to the set standards and regulations.

Results

Winter harvesting

Harvesting was conducted at night in the absence of moonlight. The pH measured in all edible offal and the carcasses is summarised in Table 2. In the livers, the pH ranged from 5.89 to 6.57, in the hearts it ranged from 5.43 to 6.89 and for kidneys the pH range was from 6.18 to 6.83. In the spleen and lungs, the pH ranged from 5.94 to 6.67 and 6.69 to 7.76, respectively. In decreasing order, the pH was higher (p < 0.05) than the reference pH of 6.0 in the lungs, kidney, spleen, liver and heart. Springbok carcasses had a pH lower (p < 0.01) than 6.0. However, there was no difference between the heart pH and the regulatory reference pH of 6.0.

Summer harvesting

Harvesting was conducted during daylight. Descriptive statistics of baseline predictor variables are shown in Table 3. In the univariate linear regression analysis, looking at the independent associations between pH and measured parameters, there were significant associations between liver pH at 4 h and liver congestion (0.08, p < 0.05) (the more the congestion, the higher the pH) and shooting to bleeding (-0.03, p < 0.05) (the longer the shooting-to-bleeding time, the lower the pH). The final multiple logistic regression model included only the variables relating to congestion in liver and liver pH at 4 h post-harvesting. Species and body condition were introduced into the multivariate model as potential confounders. In the multivariate linear regression analysis, irrespective of species, the pH of game meat 4 h after slaughter increased by 0.11 units (p < 0.05) per millilitre increase in liver congestion (Table 4). Similarly, the pH of game meat 4 h post-slaughter decreased by 0.04 units (p < 0.05) per minute increase in the shooting-to-bleeding interval (Table 5).

Discussion

One of the objectives of the study was to assess pH levels in selected chilled edible offal from springbok and other commonly harvested and exported game species so as to establish whether they meet the requirement (pH < 6.0) for safe food (Department of Agriculture 2010). The pH values of the carcasses were all below 6.0, which is consistent with requirements of the EC Regulation 206/2010 (EC 2010), which stipulates the attestation to the effect that the consignment contains boneless meat, obtained only from de-boned meat other than offal that was obtained from carcasses which have been submitted to maturation at a temperature above +2 °C for at least 24 h before the bones were removed. However, significant variations in pH ranges were observed for different offal organs originating from the renal, cardiovascular, respiratory and gastrointestinal systems. These results can be applied directly to a risk management strategy, as the study was run under ideal temperatures and humidity in the presence of competitive microorganisms under field conditions (IFT & FDA 2003; Michael et al. 2011).

Muscle pH levels of the springbok in this investigation were similar to those measured for other wild species. Deer shot in the field and deer penned without handling had muscle pH values lower than 5.74 and above 5.74, respectively, with only four of the 66 male deer transported to a slaughterhouse having a muscle pH above 6.0 (Smith & Dobson 1990). In another study, the mean pH24h measurement values were significantly higher in females than males for springbok originating from four production regions in South Africa and ranged between 5.4 and 6.3 (Hoffman, Kroucamp & Manley 2007). In a black wildebeest (Connochaetus gnou) meat study, animals harvested during the colder months of winter and autumn had a higher mean muscle pH45min, between 6.7 and 6.8, compared with animals harvested in spring at 6.2, whilst the pH24h values tend to be lower in winter, at 5.4, than in either spring or autumn at 5.6 (Hoffman, Van Schalkwyk & Muller 2009). The rate of pH decline in impala (Aepyceros melampus) carcasses harvested at night is slower compared with those harvested during the day (Hoffman, Kritzinger & Ferreira 2003). Male game animals generally tend to have a higher pH than females as a result of a more active response to disturbances, especially when harvesting occurs during the rutting season (Hoffman 2000; Lewis, Pinchin & Kestin 1997). However, sex was not taken into account during this investigation. Lymph nodes examined by Cottral, Cox and Baldwin (1960) maintained pH readings between 6.4 and 6.9, a favourable range for FMD virus survival (at 4 °C for 72 h). Liver, kidney, rumen, lymph node and blood from diseased cattle have all been shown to be highly infective and to remain so if stored frozen (Henderson & Brooksby 1948).

There are many variables that could have influenced the observed pH variations of the different organs under consideration. However, although these results are subject to potential limitations, the lack of statistically significant association between some selected variables and pH changes in this study suggests that either the factors may have a small effect that is only detectable with large data-sets and/ or the effect may have been modified by other unidentified risk-modifying factors that may be elucidated by further investigations. Factors that impact the hunting duration and time from shooting to bleeding include type of hunting terrain, species behaviour, experience and number of the hunters, and size of the harvesting camp in which the animals are found. The role of the environment, genetics and their interactions could also influence the extent of pH decrease in the offal organs (Brown, Bevis & Warriss 1990; Knee et al. 2004; Lomiwes 2008; Scanga et al. 1998; Young, Thomson, Merhtens & Loeffen 2004; Young, West, Hart & Van Otterdijk 2004; Zuliani et al. 2007). The ultimate pH of meat is not only dependent on the muscle glycogen reserves postmortem but also on other factors such as fibre type, which differs between carcasses and offal organs, and antemortem condition of the animal in terms of nutrition, health, stress and behaviour after shooting, all of which may contribute to glycogen depletion, which may cause a high ultimate muscle pH (Atanassova et al. 2008; Fink 1992; Forrest 2011; Hoffman et al. 2007; Kappelhof 1999; Smith & Dobson 1990; Van Rijswijk & Vorster 1995; Wiklund, Johansson & Malmfors 2003; Young, Thomson, Merhtens & Loeffen 2004). In this study, springbok carcasses and hearts had a pH lower than 6.0 as a result of the fact that both are muscles that have glycogen reserves and thus enter anaerobic metabolism with lactic acid as end product, an effect that could also have influenced shooting-to-bleeding time pH levels in other offal organs.

Observations on seasonal differences and effect of sex and age in ultimate carcass 24 h pH values have been reported previously (Kim et al. 2003; Van Schalkwyk 2011; Warriss 2000). Little or no glycogen is found in the lungs, but postmortem pH decline can also be slowed by the buffering effect of blood and ammonium generated by the deamination of adenosine monophosphate (Bendall & Davey 1957), therefore non-pathological haemorrhages (petechial and ecchymotic) and/or congestion sometimes observed at the postmortem inspection of the harvested springbok liver, lung and spleen may have influenced the specific organ pH post-harvest. The pH decline of muscle in a carcass deviates from a linear function because when the muscle enters rigor, hydrogen ion production decreases as the rate of anaerobic glycolysis and myosin ATPase activity decrease during muscle cooling (Bruce, Scott & Thompson 2001). The initial pH fall in muscles does not depend on the glycolytic potential, but at 5 h - 6 h and 24 h postmortem muscles with the lowest glycolytic potential have the highest pH (Maribo et al. 1998). In muscles, time taken to reach pH 6.0 from initial pH values of around 7.1 varies between 8 h and 16 h, and as low as 3.5 h in some extreme cases. Some of this variability has been attributed to different rates of cooling between muscles in the same animal (Bendall 1978). The rate of pH decrease is determined by the rate of ATP-turnover, so variability can be to the result of varying intracellular free Ca2+ levels exerting a stimulating effect on the actomyosin ATPase, with some of this extra Ca2+ arising from calcium released from the mitochondria which become anaerobic after death (Bendall 1978; Honikel, Roncales & Hamm 1983; Maribo et al. 1998). The pH readings 24 h post-slaughter that are greater than 6.5 are indicative of dark, firm and dry (DFD) carcasses, a problem frequently associated with antemortem stress. Apart from lack of visual appeal, dryness and abnormal flavour, DFD meat has increased susceptibility to bacteriological spoilage (Homer & Matthews 1998). Mean pH values have also been reported to correlate linearly with the coliform and Escherichia coli counts (Van Schalkwyk 2011).

A linear relationship of lower pH and greater bacteria reduction has been reported in treated organs as a result of induced sublethal injuries through alteration of the pH gradient of the food-borne pathogens (Dorsa, Cutter & Siragusa 1997; Flowers 2006). The effect of lactic acid on food-borne pathogens is ascribed to its ability to penetrate the cell membrane in its non-dissociated form and dissociate within the cell, thereby decreasing the pH and disturbing transmembrane proton motive force and causing an inhibition of acid sensitive enzymes (Dorsa et al. 1997; Rajkovic et al. 2010). In this study where the hearts are the only edible offal organs likely to have a pH close to or below 6.0, it can be concluded that the organs' natural pH cannot be relied on as a natural mechanism to inactivate endogenous food-borne pathogens, such as RVF virus, in some edible offal. Although international standards set a maximum pH of 6.8 as capable of inactivating RVF virus in carcasses, the primary author observed that this cannot apply to offal as the virus has been isolated in chilled livers (kept for 3-5 days at 4 °C) of healthy sheep that aborted after being transported from the farm to the abattoir. From this study, none of the offal presents a hazard if thoroughly cooked, but the consumption in some cultures of fresh blood, either alone or mixed with milk, raw or undercooked meat and organs should therefore be discouraged (Cima 2012; Corbel 2006; Countries and their Cultures 2011; Fatma & Mahdey 2010; Hall 1991; John et al. 2010; Mfinanga et al. 2003; Roux 1991).

Conclusion

As part of meat safety risk management, it may be important to consider the use of generally recognised as safe (GRAS) substances to deactivate, by pH changes, susceptible endogenous and exogenous microbial pathogens in edible offal (Berge et al. 2001; Federation of Veterinarians of Europe 2011; Food Safety and Inspection Service 2011; Harrington 2011). Treatment with GRAS should only be carried out after the official meat inspection process has been completed to prevent interference with process hygiene criteria monitoring. Further research should be conducted to establish the normal competitive microflora of the wild game offal that renders the surface of the offal susceptible to preferential growth of pathogens as a result of contamination. Further investigations with large data sets may help elucidate risk-modifying factors for pH changes in harvested wildlife. The prevalence and extent of congested carcasses and organs in harvested wildlife need to be investigated to evaluate the possible maintenance and transmission of some pathogens in different livestock systems caused by the exploding demand for livestock products.

Acknowledgements

The research and the preparation of the article were conducted using personal funds. We would like to thank Dr Nancy Ostiguy from the Department of Entomology, Pennsylvania State University for her assistance with some statistical analysis and data interpretation and the Directorate of Veterinary services for the provision of some of the equipment. Also, we would like to thank the Farmers Meat Market Abattoir HACCP team, especially Victor Mbanga, for their support in pH measurements. Thanks also to Dr E. Midzi and Dr A. Bishi for a critical review of the manuscript. Many thanks to the different game harvesting teams, especially Jan Mostert from Mos-mar game harvesting team, for their kind assistance with the provision and harvesting of some of the game species used in the study.

Competing interests

The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.

Authors' contributions

K.M. (Stellenbosch University) designed and performed all the experiments and wrote the manuscript. F.S. (University of Pretoria) made conceptual contributions, statistical analysis and interpretation of the results, whilst L.C.H. (Stellenbosch University), Y.M.H. (Directorate of Veterinary Services) and F.D. (University of West Indies) were responsible for assistance with the article review, as well as the experimental and project design.

European Commission, 2010, 'Commission Regulation (EU) No 206/2010 of 12 March 2010 laying down lists of third countries, territories or parts thereof authorized for the introduction into the European Union of certain animals and fresh meat and the veterinary certification requirements', Official Journal of the European Union L73, L146/1-121. [ Links ]

Food Safety and Inspection Service, 2011, Safe and suitable ingredients used in the production of meat, poultry and egg products, FSIS directive number 7220.1.1/4/11, United States Department of Agriculture, Food Safety and Inspection Service, Washington, DC. [ Links ]